Occurrence and Severity of Donor Lymphocyte Infusion–Associated Chronic Graft-versus-Host Disease Influence the Clinical Outcomes in Relapsed Acute Leukemia after Allogeneic Hematopoietic Stem Cell Transplantation

The aim of this study was to investigate the occurrence and severity of chemotherapy plus donor lymphocyte infusion (Chemo-DLI)-associated chronic graft-versus-host disease (cGVHD) in a consecutive cohort of patients with acute leukemia who experienced relapse after allogeneic hematopoietic stem cell transplantation (n = 104). The 5-year cumulative incidence of complete remission after Chemo-DLI was 81.0% (95% CI, 73.3% to 88.7%) and 84.6% (95% CI, 74.5% to 94.7%) in the moderate and severe cGVHD groups, respectively, which was significantly higher than that of the mild cGVHD group at 40.9% (95% CI, 29.3% to 52.5%) and non-cGVHD group at 29.2% (95% CI 23.1% to 35.3%). The cumulative incidence of nonrelapse mortality was comparable between patients with and without cGVHD. The 5-year probabilities of progression-free survival after Chemo-DLI were 42.9% (95% CI, 26.2% to 70.2%) and 34.6% (95% CI, 15.3% to 78.2%) in the moderate and severe cGVHD groups, respectively, which were both significantly higher than those of the mild cGVHD group at 9.1% (95% CI, 2.4% to 34.1%) and non-cGVHD group at 8.3% (95% CI 3.3% to 21.3%). The 5-year probabilities of overall survival after Chemo-DLI were 56.7% (95% CI, 38.9% to 82.7%) and 43.1% (95% CI, 22.1% to 84.0%) in the moderate and severe cGVHD groups, respectively, which were both significantly higher than those of the mild cGVHD group at 9.1% (95% CI 1.8% to 47.1%) and non-cGVHD group at 14.9% (95% CI, 7.3% to 30.2%). Our observations highlight the close relationship between cGVHD and immune-mediated graft-versus-leukemia (GVL) effect in patients with relapse receiving Chemo-DLI; however, mild cGVHD may not be associated with a sufficiently strong GVL effect to induce remission and improve survival.     

Self-Reactive CD4+ T Cells and B Cells in the Blood in Health and Autoimmune Disease: Increased Frequency of Thyroglobulin-Reactive Cells in Graves’ Disease

The mechanisms underlying activation of potentially self-reactive circulating B cells and T cells remain unclear. We measured the uptake of a self-antigen, thyroglobulin, by antigen presenting cells, and the subsequent proliferation of CD4⁺ T cells and B cells from healthy controls and patients with autoimmune thyroiditis. In Hashimoto's thyroiditis, B cells bound increased amounts of thyroglobulin in a complement- and autoantibody-dependent manner, and the thyroglobulin-elicited proliferation of CD4⁺ T cells and B cells was complement dependent. Increased proportions of Tg-responsive CD4⁺ T cells and B cells were found in patients with Graves’ disease. Notably, both patient groups and healthy controls exhibited higher proliferative responses to thyroglobulin than to a foreign recall antigen, tetanus toxoid. Our results suggest that self-tolerance can be broken by exposure of circulating lymphocytes to high local concentrations of self-antigen, and that complement plays a role in the maintenance of autoimmune processes, at least in Hashimoto's thyroiditis.     

Opa+ and Opa? Isolates of Neisseria meningitidis and Neisseria gonorrhoeae Induce Sustained Proliferative Responses in Human CD4+ T Cells

T cells may interact with a number of bacterial surface antigens, an encounter which has the potential to downmodulate host immune responses. Neisseria meningitidis, a human colonizer and an agent of septicemia and meningitis, expresses Opa proteins which interact with the CEACAM1 receptor expressed on activated T cells. Since CEACAM1 can act as an inhibitory receptor and T cells in subepithelial tissues may encounter whole bacteria, which often express Opa proteins in vivo, this study assessed primarily if Opa proteins expressed on meningococci affect T-cell functions. In addition, Opa-containing outer membrane vesicles (OMV) have been used as vaccine antigens, and therefore Opa⁺ and Opa⁻ OMV were also studied. While Opa⁺ bacteria adhered to CEACAM-expressing T cells, both the Opa⁺ and Opa⁻ phenotypes induced no to a small transient depression, followed by a prolonged increase in proliferation as well as cytokine production. Such responses were also observed with heat-killed bacteria or OMV. In addition, while anti-CEACAM antibodies alone inhibited proliferation, on coincubation of T cells with bacteria and the antibodies, bacterial effects predominated and were Opa independent. Thus, while Opa proteins of N. meningitidis can bind to T-cell-expressed CEACAM1, this is not sufficient to overcome the T-cell recognition of bacterial factors, which results in a proliferative and cytokine response, an observation consistent with the ability of the host to establish lasting immunity to Opa-expressing meningococci that it frequently encounters. The data also imply that Opa-proficient vaccine preparations may not necessarily inhibit T-cell functions via CEACAM1 binding.Neisseria meningitidis (meningococci) and Neisseria gonorrhoeae (gonococci), which are highly related at the genetic and antigenic levels, are human-specific mucosal bacteria capable of causing localized or systemic infection. N. meningitidis may colonize the human respiratory mucosal tissue of 3 to 30% of healthy individuals asymptomatically but, in some situations, may penetrate into deeper tissues to cause invasive diseases, such as septicemia and meningitis (5). N. gonorrhoeae may also be carried asymptomatically in a few individuals (13), but in most cases it causes localized infections of urogenital mucosa, and in a few untreated gonorrhea patients, disseminated infection may develop (20).Immune responses to mucosal bacteria are initiated at mucosa-associated lymphoid tissue, where CD4⁺ T-cell priming occurs and results in the generation of effector and memory T cells (12). Bacterial colonization, and the subsequent disease process in susceptible individuals, begins with adhesion to specific receptors on human mucosal epithelial cells. N. meningitidis and N. gonorrhoeae express colony opacity-associated (Opa) proteins in vitro and in vivo that enable them to attach to human cells. It is now well established that the major receptors targeted by the Opa proteins belong to the CEACAM (carcinoembryonic antigen-related cell adhesion molecule) family of receptors (6, 44, 45). CEACAM1 is one of several related molecules expressed on human epithelial cells, endothelial cells, and leukocytes, but CEACAM1 is the only member of the family expressed on T cells (19, 28). CEACAM1 is a transmembrane molecule with either a long (L) or a short (S) cytoplasmic tail. CEACAM1-L, with a long cytoplasmic tail, contains two tyrosine residues which form part of modified immunoreceptor tyrosine-based activation/inhibition motifs (ITAM/ITIM motifs) (16). The relative abundance of the isoforms, which may be present simultaneously in CEACAM1-expressing tissues, may dictate the signaling outcomes of CEACAM1 ligation (35).In addition, Opa structural variations may also affect bacterial specificity and affinity for distinct CEACAMs (10, 40, 44). N. meningitidis and N. gonorrhoeae possess multiple complete copies of opa genes (up to 4 and 11 genes, respectively), with the consequence that distinct isolates may express structurally variant Opa proteins. Variations within the Opa family of transmembrane proteins occur in three of the four surface-exposed loops. It has been shown for strains of distinct serogroups of N. meningitidis that these variations influence the specificity of Opa proteins for different members of the CEACAM family (10, 40). Different meningococcal isolates further possess a wide range of opa alleles, variable regions, and repertoires. Particular Opa repertoires appear to correlate with hyperinvasiveness and disease but not with the severity of meningococcal disease (4). The host cell interface where Opa proteins exert such an influence remains to be defined, and while several studies have assessed the potential of Opa proteins to influence meningococcal interactions with human epithelial cells (15, 44), a limited number of studies have examined their effect on T cells (23), and none have studied the potential of live Opa-expressing meningococci to influence T-cell functions.Previous studies have shown that a number of neisserial outer membrane proteins can modulate T-cell function. Of these, TspA (T-cell-stimulating protein A), IgA1 protease, pili, and porins can induce a proliferative response in T cells (27, 31, 34, 38). In contrast, an interaction of Opa⁺ N. gonorrhoeae with CEACAM1 inhibited immune responses of CD4⁺ T cells (2) and B cells (26). In the case of T cells, the inhibitory signal delivered by the N. gonorrhoeae Opa-CEACAM1 interaction was reported to involve the phosphotyrosine phosphatases 1 and 2 (SHP-1 and SHP-2) that interact with ITIM (2, 24). Interestingly, engagement of Opa⁺ N. gonorrhoeae with CEACAM1 on B cells occurred independently of ITIM involvement (26). Overall, the above reports highlight the following two important points: (i) with respect to cellular activation, the end product of neisseria-target cell interactions may be determined by a number of distinct bacterial and host cell characteristics; and (ii) in the context of the consequences of bacterial engagement with CEACAM1, such an interaction may not always bring into play the expected consequences of its ITIM-like motifs. Other notable observations include the following. Outer membrane vesicles (OMV) of some Opa-expressing N. meningitidis strains have been reported to inhibit CD4⁺ T-cell function (23), which is in line with CD4⁺ T-cell-inhibiting effects of Opa⁺ N. gonorrhoeae (2). However, N. meningitidis carriage is regarded as an immunizing event and has been shown to induce lasting T-cell memory (7, 8).Collectively, these previous reports highlight the need for comprehensive studies to define the consequences of meningococcal interactions with cells of the human immune system, particularly as mucosal bacteria are increasingly being shown to reside in subepithelial tissues, where they may encounter T cells. This may occur, and since no comprehensive studies are yet available on T-cell responses that may ensue upon encountering live N. meningitidis, and particularly on the influence of Opa proteins in general when presented in whole bacteria, the focus of this study was to assess the immune responses of CD4⁺ T cells to well-characterized phenotypes of live N. meningitidis and to compare these with responses to Escherichia coli cells either expressing or lacking meningococcal Opa proteins. In addition, we compared the effects of heat-killed N. meningitidis and OMV derived from a meningococcal serogroup B strain on T-cell proliferation. The latter preparations are likely to be used as vaccine preparations and therefore, in our view, warranted such studies. In order to assess the T-cell responses to other CEACAM-binding agents and to study the effects of bacterial presence when Opa-CEACAM interactions are inhibited, the studies included cross-linking antireceptor antibodies as well as a recombinant molecule, rD-7, which carries the CEACAM-binding motif of Moraxella catarrhalis UspA1 adhesin (17) and has the potential to block bacterial binding without inducing a signaling cascade in T cells because the recombinant peptide may not cross-link CEACAM1 on T cells.In our studies, frequently, but not invariably, an early Opa-independent transient decrease in T-cell proliferation was observed. This phase was followed by a profound stimulatory effect on T-cell immune functions, as assessed by proliferation assays and cytokine responses. In contrast, using anti-CEACAM1 antibodies in analogous assays, a significant inhibition of T-cell proliferation was observed. Overall, these data show that a certain surface component(s) of pathogenic Neisseria, whose precise identity remains to be determined, can exert either mild inhibitory or strong stimulatory effects on CD4⁺ T cells and that, most importantly, the latter predominate. Thus, it appears that human CD4⁺ T cells respond positively to one or more bacterial antigens to overcome any inhibition that may be induced via the engagement of CEACAM1, perhaps representing an advanced counterstrategy of the host.     

Vγ9-Vδ2+ γδ T Cells from a Patient with Felty Syndrome That Exhibit Aberrant Response to Triggering of the CD3 Molecule Can Regulate Immunoglobulin Secretion by B Cells

Expanded populations of T lymphocytes bearing γδ T cell receptors have been detected in several patients with Felty syndrome. The goal of this study was to functionally characterize these lymphocytes in a newly described patient with this disease. For this, fluorescence-activated cell sorter analysis of T cell surface antigens, proliferation, and tumor necrosis factor α enzyme-linked immunosorbent assays, as well as quantitative assays of immunoglobulins secreted by pokeweed mitogen-driven B cells were performed. The γδ cells, that expressed a CD3⁺CD4^-Vγ9^-Vδ2⁺Cδ⁺ phenotype, and constituted 60% of the peripheral blood T cells, did not proliferate after triggering with anti-CD3, but did secrete tumor necrosis factor α and the addition of these cells to pokeweed mitogen-stimulated B cells from the patient decreased their secretion of immunoglobulin M while augmenting IgG secretion. These data suggest that the expanded anergic Vγ9^-Vδ2⁺ γδ cells can play an immunoregulatory role in the patient.     

Higher CD45RA+ Regulatory T Cells in the Graft Improves Outcome in Younger Patients Undergoing T Cell–Replete Haploidentical Transplantation: Where Donor Age Matters

To understand the phenomenon of early alloreactivity (EA) in younger children undergoing post-transplantation cyclophosphamide (PTCy)-based haploidentical transplantation, we studied the graft composition and the immune reconstitution in 32 consecutive patients (aged 2 to 25 years) undergoing PTCy and T cell costimulation blockade based peripheral blood stem cell transplantation with emphasis on CD45RA⁺ subset of regulatory T cells (Tregs). All but 1 engrafted, and 14 patients experienced EA (acute graft-versus-host disease grades II to IV, n?=?8; and post-transplantation hemophagocytic syndrome, n?=?6) with a cumulative incidence of 43.7%; 42% developed mild chronic graft-versus-host disease. The overall survival was 70.2% with a nonrelapse mortality of 16.8% at a median of 19 months. Age < 10 years, donor age > 45 years, and poor recovery of Tregs correlated with EA. Not Tregs but higher CD45RA⁺ Tregs in the graft was associated with less EA (11.7% versus 32.5%, P = .0001). Higher donor age correlated with a lower CD45RA⁺ Tregs in the graft (P = .01). However, only higher CD45RA⁺ Treg percentage in the graft favorably impacted EA as well as nonrelapse mortality and overall survival. Our study demonstrates a critical role for CD45RA⁺ Tregs in determining EA and outcome after PTCy-based haploidentical peripheral blood stem cell transplantation, and the age-related physiologic decline in this population might be responsible for adverse impact of donor age.     

Distinctive Pattern of Cytokine Production and Adhesion Molecule Expression in Peripheral Blood Memory CD4+ T Cells from Patients with Active Crohn’s Disease

An expansion of both circulating and intestinal lamina propria CD4⁺CD45RO⁺ T cells has been described in patients with Crohn’s disease. We studied both the cytokine profile and the expression of adhesion molecules on this T-cell subset. Peripheral blood CD4⁺CD45RO⁺ T cells from patients with Crohn’s disease (n=45) were assessed by flow cytometry and RT-PCR methods. The cytokine profile was also measured in intestinal lamina propria from seven patients. They were classified according to the CDAI and the results were compared with those of patients with ulcerative colitis (n=21) and noninflammatory intestinal conditions (n=15), and healthy controls (n=39). The mean percentage of circulating CD4⁺CD45RO⁺ T cells producing intracellular TNF was higher in active than in inactive Crohn’s disease patients (p < 0.001), active (p = 0.49) and inactive ulcerative colitis (p = 0.019), and healthy controls (p =0. 017). TNF expression correlated with CDAI (p < 0.001). An increased expression of intracellular IL-2, IL-6, and IL-10 in active Crohn’s disease patients was also found. CD62L was downregulated in active Crohn’s disease patients while no differences were observed in CD49d and CD11a expression. Lamina propria CD4⁺CD45RO⁺ T cells from active Crohn’s disease lesions showed an increased intracellular staining of TNF, IFN-γ, and IL-10. Both peripheral and intestinal mucosa CD4⁺CD45RO⁺ T cells from active Crohn’s disease patients show an increased production of TNF. In addition, the circulating CD4⁺CD45RO⁺ T-cell subset expresses a pattern of adhesion molecules that promotes homing to extranodal lymphoid tissues. This T-cell subset may play a relevant role in the immunopathogenesis of Crohn’s disease.Dr. García de Tena and Dr. Manzano are joint first authors     

IL-35 Is Involved in the Pathogenesis of Guillain–Barré Syndrome Through Its Influence on the Function of CD4+ T Cells

CD4+ T cells and many cytokines play critical roles in the pathogenesis of Guillain–Barré Syndrome (GBS), an immune-mediated inflammatory disease. However, the role of IL-35, a novel member of the IL-12 cytokine family, in this kind of disease has not yet been elucidated. In this study, we investigated the functional changes of CD4+ T cells from GBS patients with IL-35 treatment in vitro. This study involved 21 GBS patients and an equal number of healthy controls (HCs). The results indicated that the average concentration of IL-35 in the plasma of GBS patients was lower than that of healthy controls (HCs). Increased levels of STAT1, STAT3 and STAT4 proteins and T-bet, ROR γt, IFN-γ and IL-17A mRNA were observed in CD4+ T cells from GBS patients. In contrast, the levels of STAT5 and STAT6 proteins and GATA3, Foxp3, IL-4 and TGF-β1 mRNAs were decreased in GBS patients in comparison with those of HCs. In addition, treatment of CD4+ T cells from GBS patients with IL-35 upregulated IL-35, STAT5 and STAT6 protein and T-bet, GATA3, Foxp3, IFN-γ, IL-4, IL-17A and TGF-β1 mRNA while inhibited levels of STAT3 and STAT4 protein and RORγt and IL-17A mRNA. These results indicate that IL-35 might play a potential role in GBS pathogenesis. Further studies are required in order to evaluate its role in GBS.     

IL-10/TGF-β-Treated Dendritic Cells,Pulsed with Insulin,Specifically Reduce the Response to Insulin of CD4+ Effector/Memory T Cells from Type 1 Diabetic Individuals

Introduction  

Diabetogenic autoreactive T cells with effector/memory characteristics are described in type 1 diabetes patients (T1D). Alternatively activated dendritic cells (aaDCs) have been regarded as promising tools for clinical application in autoimmune diseases (ADs), although their ability to induce antigen-specific tolerance in T cells derived from ADs has yet to be determined.     

Hydrogen-Rich Saline Ameliorates Allergic Rhinitis by Reversing the Imbalance of Th1/Th2 and Up-Regulation of CD4+CD25+Foxp3+Regulatory T Cells,Interleukin-10, and Membrane-Bound Transforming Growth Factor-β in Guinea Pigs

It is well known that CD4+CD25+Foxp3+Treg cells play an important role in the development of allergic rhinitis (AR); the defect of cell numbers and functions contribute to AR. Hydrogen has been proven effective in alleviating symptoms of AR. We herein aim to verify the protective effects of hydrogen on CD4+CD25+Foxp3+Treg cells in guinea pigs with AR and to explore the effect of hydrogen-rich saline (HRS) on CD4+CD25+Foxp3+Treg cells in animals with AR and investigate the underlying anti-inflammatory mechanism. Eighteen guinea pigs were randomly divided into three groups (control group/AR group/AR-HRS group). The guinea pigs were injected with hydrogen-rich saline (AR-HRS group) for 10 days after sensitization. The control group was injected with an equal volume of normal saline. The number of sneezes, degree of runny nose, and nasal-rubbing movements were scored. Peripheral blood eosinophil count was recorded. The proportions of Th1/Th2 of the peripheral blood and the CD4+CD25+Foxp3+T cells in the CD4+T cells of the spleen and peripheral blood were determined by flow cytometry. The content of interleukin (IL)-10 and transforming growth factor (TGF)-β in the serum was detected by enzyme-linked immunosorbent assay (ELISA). The protein and mRNA expression of Foxp3, IL-10, and TGF-β were determined by Western blot, immunofluorescence, and real-time PCR analysis, respectively. Scores of symptoms, number of eosinophils,and nasal mucosa damage were dramatically reduced after HRS treatment. HRS increased the expression of Foxp3, IL-10, TGF-β, and number of CD4+CD25+Foxp3+Treg cells, which were reduced in AR. HRS also revised the dysregulation of Th1/Th2 balance. Both the number and biological activity of CD4+CD25+Foxp3+Treg cells increased with up-regulation of Th1/Th2 after HRS administration. HRS could play a protective role in attenuating AR through improving the proportion and functions of CD4+CD25+Foxp3+Treg cells.